The third component of complement, C3, is an important participant in immune surveillance and immune response pathways. Activation of the complement system by the classical or alternative pathways results in a and controlled fragmentation of the C3 molecule. Interactions of C3 fragments with cell surface receptors serve to modulate complement-dependent granulocyte and lymphocyte functions. The elucidation of molecular features germane to these complement functions will require (1) further characterization of the structure of the individual C3 subdomains involved, and (2) an evaluation of the physiochemical basis for their binding properties. At the structural level, our objective will be to complete the primary structure of C3 both by limited protein sequence analysis of the major cyanogen bromide fragments and also by full sequence analysis of cloned cDNA encoding the C3 molecule. Once human C3 cDNA clones are identified in a human cDNA library, the emphasis will shift from mouse to human. This combined approach, whereby limited protein sequence data is primarily used to confirm the isolation of specific cDNA clones and to insure proper reading frame assignments, has the potential to shorten by several years the time required to establish the complete primary structure of C3. Furthermore, the structure of C3 from two species (mouse and human) will be compared in order to determine the structural correlates for specific functional domains of the molecule. We have recently identified a unique thiolester bond in C3 which is strictly required for the hemolytic function of this molecule. We propose to delineate the biosynthetic pathway(s) for formation of the thiolester bond, determine the mechanism whereby the acyl component of the thiolester is transferred to acceptor molecules with formation of a covalent bond, and study further examples of thiolester-containing proteins of eukarocyte origin. The major transforming antigen of simian virus 40 is a major candidate. Stable cell receptor binding sites have been shown to be associated with several subdomain structures of C3 (i.e. C3b, iC3b, C3d, and C3c). We propose to develop direct binding assays for the C3 receptors, CR2 and CR3, and identify, by inhibition studies, peptides from the major subdomains of C3 which retain specific receptor binding activity. These peptides may have specific application as inhibitors of in vivo C3 receptor interactions, have the potential of offering a new class of inhibitors to the processes involved in inflammatory reactions.